Project Summary: Maze I ! Down syndrome (DS) is the most common genetic cause of intellectual disability worldwide resulting from triplication of chromosome 21 (HSA21) in humans. Despite much progress in understanding the genetics of DS, the genes encoded on HSA21?and beyond?that directly contribute to intellectual disability, as well as their associated molecular mechanisms, remain incompletely understood. BRWD1 is an uncharacterized, brain enriched chromatin `reader' protein encoded within the DS critical region 2 on HSA21. BRWD1 is believed to act as a transcriptional activator via its proposed interactions with the SWI/SNF chromatin remodeling complex; however, its roles in the contexts of neurodevelopment and in DS have yet to be studied. Consistent with previous gene expression analyses in human DS brain, we observe that Brwd1 expression is significantly elevated in trisomic mice, as well as in neurons derived from hiPSCs from DS subjects. Select histone posttranslational modifications (PTMs), some of which we have already shown to directly interact with BRWD1, are also altered in their expression. We have demonstrated that genetic re-normalization of Brwd1 expression in Ts65Dn mice significantly rescues transcriptional dysplasticity associated with hippocampal LTP and cognitive deficits in trisomic animals. Furthermore, exogenous Brwd1 overexpression?via viral-mediated transduction of adult dorsal hippocampal neurons in wildtype animals?also leads to deficits in both contextual and spatial memory. It is our hypothesis that BRWD1 trisomy in DS brain results in aberrant interactions between BRWD1 and the epigenetic landscape, thereby contributing to global gene expression abnormalities and cognitive deficits. We seek to test this hypothesis in the following independent, yet synergistic Aims: 1) Examine the relationship between Brwd1 trisomy and chromatin dysfunction in DS-like brain, with an overarching emphasis on a) unbiased assessments of histone PTM expression in DS-like neurons and glia (+/? Brwd1 rescue) using cell sorting methodologies coupled to histone PTM mass spectrometry, and b) delineation of direct interactions between BRWD1 and altered histone PTMs using highly novel `designer' nucleosome-based assays and in vivo validations. 2) Investigate the impact of Brwd1 triplication on gene expression in DS-like brain. We aim to perform unbiased, epigenome-wide analyses of Brwd1 enrichment?along with associated histone PTMs?in Ts65Dn brain, followed by extensive comparisons with gene expression profiles obtained using cell-type specific RNA-seq. CRISPR-based manipulations will subsequently be performed to directly assess Brwd1 target gene contributions to observed behavioral abnormalities. 3) Explore the impact of Brwd1 triplication on recruitment/activity of specific SWI/SNF chromatin remodeling complexes in Ts65Dn mouse brain using biochemical and genome-wide assessments. These experiments will allow us to dissect the functional consequences of abnormal patterns of chromatin regulation in DS brain and will aid in our understanding of the neuroepigenetic processes associated with brain plasticity.